The translation of the viral genome begins immediately after coronavirus (CoV) infection to produce two large polyproteins that are processed into 15 or 16 mature, nonstructural proteins. Most of these proteins are involved in viral RNA synthesis, and some have other biological functions. The nsp1 protein of betaCoVs, which includes SARS-CoV (SCoV), bat CoVs, and mouse hepatitis virus (MHV), and of alphaCoVs, which includes transmissible gastroenteritis virus (TGEV) and human CoV 229E, inhibits host gene expression. As past studies have shown that viral proteins that inhibit host gene expression are major virulence factors, nsp1 proteins of CoVs most probably play a critical role in CoV pathogenesis. Consistent with this notion, MHV nsp1 is a major virulence factor, and SCoV nsp1 inhibits the production of type I interferon and interferon-stimulated genes in infected cells. Nsp1 proteins of different CoVs share a common biological function to inhibit host gene expression but use different strategies to exert this function. SCoV nsp1 uses a novel, two-pronged strategy to inhibit host protein synthesis/gene expression. SCoV nsp1 binds to the 40S ribosomal subunit to inhibit mRNA translation and also induces a template-dependent endonucleolytic mRNA cleavage. In contrast to SCoV nsp1, TGEV nsp1 employs a different strategy to inhibit mRNA translation, as it is unable to bind the 40S ribosomal subunit or promote host mRNA degradation. This application aims to delineate the novel mechanisms of CoV nsp1-induced inhibition of gene expression by using SCoV and TGEV nsp1 proteins as model systems. We will uncover the mechanism of SCoV nsp1-induced template-dependent mRNA cleavage. We will also clarify the different mechanisms of SCoV and TGEV nsp1-induced translation inhibition and reveal the strategy that allows robust viral gene expression in SCoV-infected cells under conditions of nsp1-induced translation inhibition. The proposed studies will provide a foundation for understanding the modulation of host gene expression by CoV, expand our knowledge of CoV pathogenicity at the molecular level, and potentially identify a novel mechanism for the regulation of eukaryotic gene expression. PUBLIC HEALTH RELEVANCE: The proposed studies will delineate the novel mechanisms of coronavirus nsp1-induced inhibition of gene expression. These studies will provide a foundation for understanding the modulation of host gene expression by coronaviruses, expand our knowledge of coronavirus pathogenicity, and potentially identify a novel mechanism for the regulation of eukaryotic gene expression.